Over the last ten years many reports have provided compelling evidence for a competitively priced bio-based products industry that will replace much of the petrochemical industry. In order to reduce U.S. reliance on foreign oil, there is an increasing interest in generating commodity and fine chemicals from widely available U.S. renewable resources through fermentation. In the last five years, billions of dollars have been invested in commercializing the microbial production of several chemicals, including lactic acid, 1,3-propanediol and 3-hydroxypropanoic acid. In concert with this increased interest in crop-derived biochemicals, molecular biology has become a standard tool which engineers and applied scientists use routinely to aid the rational and targeted alteration of metabolism.
The efficient use of agricultural biomass for the production of any biochemical, however, is problematic. Technical challenges to be overcome in order for bio-based industrial products to be cost-competitive include finding new technology and reducing the cost of technology for converting biomass into desired bio-based industrial products. Research resulting in cost-effective technology to overcome the recalcitrance of cellulosic biomass would allow biorefineries to produce fuels and bulk chemicals on a very large scale.
The untapped sources of biomass are largely lignocellulosic in nature. One promising use of lignocellulose for liquid fuel is in the microbial production of ethanol. Unfortunately, when broken down into constituents, a very complex mixture remains. This mixture contains sugars which individually but not collectively are suitable for fermentation, and the mixture also contains inhibitors.
Because the unit value of chemical products derived from biomass (e.g., ethanol) is generally low while the potential market is large, the economic viability of such processes depends on the yield and productivity. Yield is the quantity of product formed per mass of material input, while productivity is the rate at which the product is generated. Achieving high yield demands that all biomass components be converted, while high productivity requires that the complex conversions be accomplished quickly.
The conversion of lignocellulosic biomass to any useful fermentation product follows a series of general process operations which include identifying the biomass, harvesting it, various separation/extraction steps, pretreatments, conversion and subsequent purification steps. Although the particular substrate and chemical product determine the details of each operation, the complex structure of biomass invariably necessitates that its components be broken down by various depolymerization strategies.
Fermentation is the biological process in which sugar substrates, such as glucose and xylose, are converted into fermentation products, such as ethanol. While the term fermentation is usually reserved for anaerobic processes, analogous microbial processes similarly convert sugar substrates into products under a controlled aerobic environment or under conditions of partial oxygenation. One major limitation is that, regardless of a microorganism's ability to metabolize multiple substrates, a single substrate persistently remains the preferred substrate and the consumption of the sugars is asynchronous. Invariably, a multitude of substrates remains long after the preferred substrate has been metabolized. Numerous attempts to engineer microorganisms capable of equally metabolizing more than one substrate have been made (Dien et al., 2002, J. Industr. Micro. Biotech. 29:221; Sedlak et al., 2003, Enzyme Micro. Technol. 33:19; Chandrakant and Bisaria, 2000, Appl. Micro. Biotechnol. 53:301; Kuyper et al., 2005, FEMS Yeast Res. 5:925; Zhang et al., 1995, Science 267:240). However, none has prevented the polyauxic behavior of linearly metabolizing one sugar at a time when metabolizing sugar mixtures (i.e., first glucose consumption, then xylose, etc.).
A single microorganism alone is unable to convert multiple sugars simultaneously. Instead, any given microorganism has a complex regulatory network which forces sugars to be metabolized sequentially. This sequential nature invariably reduces the overall rate of a fermentation process to generate the desired product.
Hydrolysis of the lignocellulosic biomass releases variety of sugars, including hexoses (e.g., glucose, mannose), pentoses (e.g., xylose, arabinose), and oligosaccharides, that are released by the hydrolysis of lignocellulosic biomass, and no single microorganism is capable of fermenting all these sugars. Many ethanologenic microbes, including yeast, prefer to use glucose as a substrate. Even when yeast cells are modified genetically to use xylose, they ferment all glucose before switching to the much slower xylose fermentation. Conversion rates can vary greatly depending on such factors as the type of sugar substrate being fermented, environmental conditions (e.g., pH, temperature), and the concentrations of certain products from other metabolic pathways.
The fraction of pentose sugars which compose biomass can significant; for example, 12% pentose sugars have been reported for Pinus spp. and 26% for Populus spp. (Saddler and Mackie, 1990, Biomass 22:293). Acid/enzymatic hydrolysis of agricultural materials also generates a significant fraction of pentoses. For example, hydrolysis of peanut hulls results in a mixture containing 44% pentoses (Chandrakant and Bisaria, 2000, Appl. Micro. Biotechnol. 53:301). In order to achieve high yields and productivities, both pentose and hexose fractions must be fully and efficiently utilized. Complicating the matter is the fact that hydrolysis also leads to the formation of acetic acid, which is a known inhibitor to any of the microorganisms that might be used to ferment these sugars into products such as ethanol.
The efficient conversion of both pentoses and hexoses is a significant hurdle to the economic utilization of biomass hydrolysates for the generation of any fermentation product. The central problem is that either the microorganism used to metabolize the sugars in the mixture consumes the sugar constituents sequentially (e.g., first glucose and then xylose) or the organism is unable to utilize the pentose at all (as is the case with the yeast, Saccharomyces cerevisiae). In a recent comprehensive review Zaldivar et al. succinctly conclude “the lack of a microorganism able to ferment efficiently all sugars released by hydrolysis from lignocellulosic materials has been one of the main factors preventing utilization of lignocellulose” (Zaldivar et al., 2001, Appl. Microbiol. Biotechnol. 56:17).
Current strategies have focused on the development of a single organism engineered to metabolize both hexoses and pentoses, a single organism that “can do it all.” For example, the common yeast Saccharomyces cerevisiae, the most widely used organism for ethanol production from starch-based crops, has been genetically modified to metabolize xylose as well as its native substrate glucose. Genes encoding xylose reductase, xylitol dehydrogenase and xylulokinase fused to glycolytic promoters have been successfully integrated into the yeast chromosome (Ho et al., 1998, Appl. Env. Micro. 64:1852.; Sedlak et al., 2003, Enzyme Micro. Technol. 33:19.). In another study, S. cerevisiae genetically engineered to contain genes to metabolize xylose still consumed less than 25% of the xylose when glucose was depleted (Sedlak et al., 2003, Enzyme Micro. Technol. 33:19.). Even when xylose isomerase activity was added to S. cerevisiae to convert xylose to glucose extracellularly, 75% of the xylose still remained after the glucose was completely consumed (Chandrakant and Bisaria, 2000, Appl. Micro. Biotechnol. 53:301.).
Bacteria are also frequently used for fermentation processes, but are unable to efficiently metabolize sugar mixtures. In many bacteria, the metabolism of glucose prevents efficient xylose consumption and as a result many researchers have attempted to improve the efficiency of xylose consumption. Introducing a mutation into the ptsG gene of Escherichia coli can reduce glucose-mediated repression of xylose consumption (Dien et al., 2002, J. Industr. Micro. Biotech. 29:221.). For example, in batch culture with the ethanologenic E. coli strain K011 grown on hemicellulose hydrolysate, for example, only 11% of the xylose was consumed after 24 h, while 80% of the glucose was consumed (Barbosa et al., 1992, Appl. Env. Micro. 58:1382.). Removal of the ptsG improved xylose consumption in the presence of glucose, but still 40% of the xylose remained when the glucose is depleted (Dien et al., 2002, J. Industr. Micro. Biotech. 29:221.).
Approaches using “evolutionary engineering” have also significantly improved the rate of xylose consumption (Kuyper et al., 2005, FEMS Yeast Res. 5:925), but have not prevented the diauxic behavior when using sugar mixtures (i.e., first glucose consumption, then xylose). Likewise, introduction of genes involved in the xylose metabolism pathway into Zymomonas mobilis does not prevent it from consuming xylose much more slowly than glucose (Zhang et al., 1995, Science 267:240). Because both sugars are not consumed effectively in any of these single-organism processes, the productivity of the process is suboptimal.
Strategies which require a single organism to convert xylose and glucose suffer from several limitations. First, as noted above, the consumption of the sugars is asynchronous. Despite the presence of the genetic apparatus to consume both sugars, glucose remains the preferred substrate, and xylose invariably remains when glucose has been consumed. This asynchronicity particularly influences a single microorganism's ability to cope well with a real hydrolysate having a temporally varying concentration of each sugar. Faced with a time-varying stream, yet using a single organism which has a limited ability to adjust its ratio of glucose and xylose consumption rates, the process will invariably lead to one of the sugars not being effectively consumed. It is not currently possible for one organism to “adjust” its rate of consumption to two substrates in order to match the concentrations encountered in a real process.
A second shortcoming is that a single microorganism strain, which has been engineered to consume both glucose and xylose, tends to be unstable. A study using a chemostat demonstrated that the presence of both sugars caused an increase over time in the by-product (and inhibitor) acetic acid, which ultimately led to a 20% decrease in ethanol yield (Dumsday et al., 1999, J. Indust. Micro. Biotechnol. 23:701).
This highlights another complication: hydrolysis of real biomass typically generates compounds which inhibit the subsequent conversion of sugars by fermentation. For example, xylose is acetylated in lignocellulose (Timell, 1967, Wood Sci. Technol. 1:45; Chesson et al., 1983, J. Sci. Food Agric. 34:1330), and therefore acetic acid is an unavoidable product of hemicellulose depolymerization. Although the inhibitory effect of acetic acid depends on the strain and process, an acetic acid concentration of only 0.08% has been demonstrated to inhibit a subsequent fermentation to generate ethanol (van Zyl et al., 1991, Enzyme Micro. Technol. 13:82). Generally acetic acid inhibits xylose conversion more than it affects glucose conversion. In S. cerevisiae engineered to metabolize xylose, for example, acetic acid reduced ethanol yield from xylose by 50% (Helie et al., 2003, Enzyme Microbial Technol. 33:786). Acetic acid itself, and not merely the pH, causes the inhibition. Therefore, base neutralization traditionally applied to acid treated lignocellulosic hydrolysates does not eliminate the inhibitory affects of acetic acid. Previous approaches to convert this mixture of sugars and inhibitors have not been able to achieve the rate of conversion necessary to make the process economically viable. A novel approach that successfully overcomes the inability to convert a mixture of sugars and inhibitors and increases the yields of fermentation product per amount of biomass would represent a significant and long awaited advance in the field.